The invention relates to novel invertebrate TWIK channel nucleic acid and polypeptide sequences and their uses in genetic screens and compound screening.
Potassium channels are present in virtually all living cells, and are the most diverse class of ion channels. They conduct the flux of potassium ions through the membrane, and in doing so, are involved in the control of numerous cellular functions, such as neuronal firing, muscle contraction, volume regulation, cellular proliferation, and hormone secretion (Rudy, Neuroscience (1988) 25:729-749; Hille in: ionic Channels in Excitable Membranes, 2nd Ed. (1992) Sinauer Associates, Inc., Sunderland, Mass.). Two characteristic features of this class of channels are pore-forming (P) domains that have a conserved sequence motif, and at least two transmembrane (TM) domains. It is believed that four P domains contribute to the formation of a functional potassium-conducting pore (MacKinnon, Nature (1991) 350:232-235; Yang et al., Neuron (1995) 15:1441-1447; Doyle et al., Science (1998) 280:69-77).
There are three structural classes of potassium channels, based on their protein encoding subunits, and defined by the number of TM and P domains: 1) voltage-gated, Shaker-like outward rectifier K+ channels are characterized by the presence of six TM domains and one P domain; 2) inward rectifier, G-protein-coupled K+ channels are characterized by 2 TM domains and one P domain; and 3) tandem pore domain weak inward rectifying K+ (TWIK) channels, depicted in FIG. 1, are characterized by the presence of four TM domains and two P domains. The P domains of the TWIK channels are separated by the second and third TM domains. Although all members of this family have a conserved core region between the first and fourth TM domains, thexe2x80x94and C-terminal domains are quite diverse. The TWIK-related channels, TREK-1, TASK (also called cTBAK-1), and TRAAK exhibit the same overall structure, despite their low similarity at the amino acid level (Fink et al., EMBO J (1996) 15:6854-6862; Duprat et al., EMBO J. (1997) 16:5464-5471; Fink et al., EMBO J.(1998) 17:3297-3308; Leonoudakis et al., J. Neurosci. (1998) 18:868-877; Kim et al., Circ. Res. (1998) 82:513-518). Some TWIK family members are known to dimerize through the presence of a disulfide bridge (Lesage et al., EMBO J. (1996) 15:6400-6407), and seem to be involved in the generation and modulation of the resting potential of many cell types (Lesage and Lazdunski in: Potassium Ion Channels: Molecular Structure, Function, and Diseases (1998), Kurachi et al., eds., Academic Press, San Diego, Calif.). TWIK channels are widespread in mammals (Lesage et al., EMBO J. (1996) 15:1004-1011; Chavez et al., J. Biol. Chem. (1999) 274:7887-7892) and in Caenorhabditis elegans (hereinafter xe2x80x9cC. elegansxe2x80x9d; Wei et al., Neuropharmacology (1996) 35:805-829). To date, however, only one TWIK channel from Drosophila melanogaster has been reported (Goldstein et al., PNAS (1996) 93:13256-13261; U.S. Pat. No. 5,559,026); and there have been no further reports of TWIK channels in other insect species.
Pesticide development has traditionally focused on the chemical and physical properties of the pesticide itself, a relatively time-consuming and expensive process. As a consequence, efforts have been concentrated on the modification of pre-existing, well-validated compounds, rather than on the development of new pesticides. A promising alternative is to identify and validate biological targets against which potential ligands can be screened (Margolis and Duyk, Nature Biotech. (1998) 16:311). Production of new compounds that are safer, selective, and more efficient can be implemented using target-based discovery approaches. Further, identifying molecular diversity addressing such targets may be exploited via combinatorial chemistry and high-throughput screening. High-throughput assays can be run rapidly and inexpensively and, due to their scale, allow access to the structural variety granted by combinatorial chemistry. In addition, potential lead compounds can be directly counter-screened on the same target cloned from human or beneficial insect sources to exclude broad spectrum toxins. The essential functions of target genes in insects and nematodes may be tested directly using powerful genetic methods, eliminating the costly uncertainty of whether or not a specific gene or biochemical activity might be a pesticide target. The phenotypic consequence of genetically modulating target gene activity serves as a surrogate for chemical inhibition or activation of a protein target. Thus, genes that kill the organism when over-expressed or knocked out represent first-stage validated targets. To identify compounds that have the same effect on the organism, high-throughput screening assays are established to test compounds for their ability to interfere in vitro with the normal activity of the target. Biological definition of targets provides the opportunity to optimize chemistry around validated targets. Ion channels are validated targets of insecticide action. A significant portion of commercial insecticides are targeted towards GABA-gated chloride channels and voltage-gated sodium channels. Potassium channels, as a diverse group, have yet to be fully exploited as targets for insecticidal agents.
Potassium ion channels are involved in numerous cellular functions in a variety of cell types, and recent advances in genomics and physiology have identified several potassium channels that are involved in human diseases (Doyle et al., Trends. Genet. (1998) 14:92-98). In particular, members of the TWIK family show strong expression in the brain and the heart, followed by expression in the kidney and the muscle (Lesage et al., supra), and thus, could be implicated in epilepsy, cardiac pathologies (arrhythmias), vascular defects, neurodegenerative disorders, endocrinopathies, hormone secretion and muscular defects, and genetic diseases. The growing body of information regarding the modular subunits and the high-resolution structural of these channels features (Doyle et al., Science (1998) 280:69-77) provide critical information for validation of potassium channels as drug targets. The identification of novel TWIK orthologues in model organisms such as Drosophila melanogaster and other insect species would provide tools for genetic and molecular study and validation of these molecules as potential pesticide or pharmaceutical targets.
The present invention relates to the identification and characterization of novel TWIK channels in Drosophila melanogaster and Leptinotarsa decemlineata. Isolated nucleic acid molecules are provided that encode TWIK channel proteins or novel fragments or derivatives thereof. Vectors and host cells comprising the TWIK channel nucleic acid molecules are also described, as well as metazoan invertebrate organisms (e.g. insects, coelomates and pseudocoelomates) that are genetically modified to express or mis-express a TWIK channel protein.
An important utility of the novel TWIK channel nucleic acids and proteins of the present invention is that they can be used in screening assays to identify candidate compounds which are potential pesticidal agents or therapeutics that interact with TWIK channel proteins. Such assays typically comprise contacting a TWIK channel protein or fragment with one or more candidate molecules, and detecting any interaction between the candidate compound and the TWIK channel protein. The assays may comprise administering the candidate molecules to cultured host cells that have been genetically engineered to express the TWIK channel proteins, or alternatively, administering the candidate compound to a metazoan invertebrate organism that has been genetically engineered to express a TWIK channel protein.
The genetically engineered metazoan invertebrate animals of the invention can also be used in methods for studying TWIK channel activity. These methods typically involve detecting the phenotype caused by the expression or mis-expression of the TWIK channel protein. The methods may additionally comprise observing a second animal that has the same genetic modification as the first but additionally comprises a mutation in a gene of interest. Differences, if any, between the phenotypes of the two animals identifies the gene of interest as capable of modifying the function of the gene encoding the TWIK channel protein.